The Type-it Microsatellite PCR Kit is based on highly specific HotStarTaq Plus DNA Polymerase and a patented buffer system, both of which enable reliable multiplex PCR-based microsatellite analysis without optimization. The combination of all components provided in the master mix and the specialized formulation result in highly specific amplification of all loci in parallel. The kit is dedicated to fast and reliable microsatellite analysis by multiplex PCR. Optimized protocols are also provided to enable subsequent analysis either by high-resolution capillary sequencing or by using other electrophoresis instruments.

Only 2 out of 4 channels (FAM and PET) of a 3730 xl Capillary Sequencer (ABI), representing 6 of 13 analyzed STR loci are shown. [A] Using the Type-it Microsatellite PCR Kit under standard conditions, all 6 STR loci are reliably amplified. [B] Despite the increased number of cycles and preoptimized PCR conditions for the hot-start method of Supplier A — compared with 25 cycles and standard conditions for the Type-it Microsatellite Kit — several specific peaks are missing and nonspecific signals are observed (arrows).|The Type-it Microsatellite PCR Kit is based on proprietary QIAGEN multiplex technology and provides a simple procedure for reliable genotyping results. In contrast to current methods, the need for optimization of PCR parameters such as Mg2+ concentration and cycling conditions is virtually eliminated.|Unique Type-it Microsatellite PCR Buffer promotes stable and efficient annealing. [A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, synthetic Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.|

The Type-it Microsatellite PCR Kit outperformed kits tested from other suppliers and ensures reliable microsatellite analysis. The kit is specifically developed and functionally validated for multiplex PCR-based analysis of microsatellites or minisatellites such as STRs or VNTRs used, for example, in relationship analysis or population genetics (see figure "Reliable 13-plex STR analysis without the need for optimization").

Principle

The Type-it Microsatellite PCR Kit provides fast and reliable genotyping analysis of humans, animals, plants, and bacteria using microsatellites, STR, or VNTR markers, without the need for lengthy optimization procedures. The Type-it Microsatellite PCR Kit is based on proprietary QIAGEN multiplex technology and includes highly specific HotStarTaq Plus DNA Polymerase, a chemically modified hot-start enzyme, and a specially developed multiplex buffer system including Factor MP. Together, these components ensure high yields and enable reliable multiplex amplification of all microsatellite loci of interest. The kit includes dedicated protocols for the amplification of microsatellites or STRs, as well as step-by-step advice on template amounts, cycle numbers, and PCR conditions and instrument details for different downstream analysis platforms such as agarose gels, capillary sequencers, the Agilent Bioanalyzer, and the QIAxcel Advanced System.

Type-it Microsatellite PCR Buffer

Unique Type-it Microsatellite PCR Buffer facilitates the amplification of multiple PCR products. In contrast to conventional PCR reagents, the Type-it Microsatellite PCR Buffer contains a specially developed, balanced combination of salts and additives to ensure comparable efficiencies for annealing and extension of all primers in the reaction. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR buffer provides stringent primer-annealing conditions over a wider range of temperatures and Mg2+ concentrations than conventional PCR buffers. The need to optimize PCR by varying the annealing temperature or the Mg2+ concentration is dramatically reduced, or often not required. Commonly employed optimization procedures for multiplex PCR are virtually eliminated. The buffer also contains the synthetic Factor MP (see figure "Stable and efficient annealing"), which allows efficient primer annealing and extension, irrespective of primer sequence. Factor MP increases the local concentration of primers at the DNA template and stabilizes specifically bound primers.

Q-Solution

The Type-it Microsatellite PCR Kit includes Q-Solution. This innovative PCR additive facilitates amplification of difficult templates by modifying the melting behavior of DNA. Use of this unique reagent will often enable or improve suboptimal PCR. Unlike DMSO and other PCR additives, Q-Solution is used at a defined working concentration with any primer-template system and is not toxic.

Procedure

Dedicated, application-specific protocols, optimized for analysis of microsatellites on high-resolution capillary sequencers, are included with the Type-it Microsatellite PCR Kit to ensure reliable results for routine analysis, as well as for the establishment of new assays. Reactions can be set up at room temperature, ensuring greater convenience and ease of use.

Fast and simple way to reliable genotyping results

The Type-it Microsatellite PCR Kit enables fast and easy multiplex assay development for accurate and reproducible genotyping results. Whatever the application — microsatellite, STR, SSR, or VNTR analysis — just add the template and primers, and start the thermal-cycler program according to the optimized protocol. The reaction mixture contains all of the reagents required for microsatellite-specific multiplex PCR and enables accurate results without the need for lengthy optimization procedures compared to current methods (see figure "Successful multiplex PCR-based genotyping analysis").

Optimized for use with capillary sequencers

Microsatellite analysis on high-resolution detection systems such as capillary sequencers is often associated with the problem of uneven product yield and huge intensity differences of fluorescent signals. The Type-it Microsatellite PCR Kit overcomes this limitation and ensures high product yields for all amplicons in a multiplex experiment. The kit includes optimized protocols for use with fluorescent primers and outperforms enzyme solutions from other suppliers (see figure "Reliable 13-plex STR analysis without the need for optimization").

Applications

The Type-it Microsatellite PCR Kit is used to analyze microsatellites, STRs (short tandem repeats), SSRs (simple sequence repeats), and VNTRs (variable number of tandem repeats) and can be used in diverse research fields such as:

Species or individual identification

Analysis of relationship

Microsatellite instability

Population genetics

Feature

Specifications

Applications

Multiplex PCR, Microsatellites, STRs, VNTRs, SSRs

Mastermix

Yes

Product use

Functionally validated and developed for reliable Microsatellite Analysis